Lens Junctions Research Articles

Biochemical evidence for adhesion-promoting role of major intrinsic protein isolated from both normal and cataractous human lenses

In this study, we tested the adhesion-promoting role of major intrinsic protein from both normal human (cadaver) and senile cataractous lenses. Junctional membrane solubilized proteins and pure major intrinsic protein obtained from both type of lenses were reconstituted in neutral phosphatidylcholine liposomes. The interaction of these liposomes with phosphatidylserine vesicles was studied by resonance energy transfer. Our results show that normal human lens junction solubilized proteins and pure major intrinsic protein isolated from them promote adhesion. No quenching effect was observed when major intrinsic protein was omitted in the vesicle reconstitution, no other intrinsic protein of normal human junctional membrane provoked the adhesive effect. In contrast, major intrinsic protein isolated from human senile cataractous lens fails to induce adhesion. The proteolytic cleavages that in vitro originate major intrinsic protein 22,000 Da did not blunt its adhesive capability, suggesting that the proteolytic modifications that major intrinsic protein undergoes in senile cataract were not related with the incompetence of cataractous lens junctions to induce adhesion. Cataractous lens junctional membranes showed protein aggregates. These membranes were treated with sodium hydroxide and reconstituted into liposomes. The sodium hydroxide treatment removed the protein aggregates and restored the adhesive capability. Furthermore, the supernatant obtained after the sodium hydroxide treatment of cataractous junctional membranes, inhibited the adhesive effect of vesicles reconstituted with bovine solubilized proteins. These experiments prove that the failure to induce adhesion of human senile cataractous lens junction proteins is due to the interaction with protein aggregates, which can be removed by sodium hydroxide.

The gap junction proteins: vive la différence!

AbstractThe intercellular junctions connecting the cytoplasms of fibre cells in the mammalian lens have until recently been regarded as a class of junction which is fundamentally different from that of the gap junctions in other organs. Recent observations, however, suggest that the lens junctions fit protein topology predictions common for all gap junctions. While the homologous peptide portions are predicted to form the channels, the divergent peptide portions of the gap junction polypeptides may adapt channel activity to the special tissue requirements.

Junctions between lens cells in differentiating cultures: Structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated “lentoids” are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eightfold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.

Phosphorylation of lens intrinsic membrane proteins by protein kinase C

Two intrinsic proteins of bovine lens membranes with apparent relative molecular masses (Mr, app) of 26,000 and 18,000 were phosphorylated in intact membranes by protein kinase C prepared from either bovine brain or lens. The kinase preparations exhibited histone H1 phosphorylation dependent on calcium and phospholipid but not on cAMP. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the lens membranes showed a major band at Mr, app = 26,000 (identified as MP26, the main intrinsic protein of lens fiber cells), an intermediate band at Mr, app = 18,000 and several minor bands. Autoradiography of complete assay mixture containing protein kinase C, calcium, magnesium and [gamma-32P]ATP showed major bands at Mr, app = 18,000 and 26,000. Several lines of evidence indicated that the label at Mr, app = 26,000 was associated with MP26, a protein which has been found in lens junctions and which may form cell-cell channels. Treatment of the phosphorylated membranes with chymotrypsin and V8 protease cleaved the major band at Mr, app = 26,000 to fragments of Mr, app .= 22,000 and 24,000. Label was not detected in the resulting Mr, app = 22,000 peptide, but the Mr, app = 24,000 peptide was found to be labeled. Phosphoamino acid analysis of MP26 indicated that approximately 75% of the label was on phosphoserine and 25% was on phosphothreonine. No label was found on phosphotyrosine. These results differ from those reported for cAMP-dependent phosphorylation of lens proteins. Phosphorylation by protein kinase C may account for some of the labeling of MP26 detected in vivo.

Calcium-binding lens membrane proteins.

Calcium-binding membrane-bound proteins are present in the vertebrate eye lens. Among these proteins are a distinct group of immunologically related extrinsic EDTA-extractable proteins (EEP) and calmodulin. The EEP proteins contain calcium-binding sites with a total capacity of 25 mol Ca2+ per mol protein. This high calcium-binding capacity of EEP points to a function of these proteins as intracellular calcium store in the lens. However, EEP undergoes a conformational change upon calcium binding, indicating that these proteins may be involved in the regulation of calcium-dependent cellular processes in the lens. One of these processes is the action of communicating lens fiber junctions, which contain EEP as a main protein component. In addition to EEP, another calcium-binding protein in lens, calmodulin, probably functions as mediator of calcium in the regulation of the structure and function of lens junctions. Like other vertebrate calmodulins, lens calmodulin shows a calcium-dependent mobility shift on SDS-polyacrylamide gels and forms immune complexes with antiserum raised against vertebrate calmodulin. Lens calmodulin binds to the junction proteins MIP (main intrinsic protein, MW 26 Kdalton) and a 17.5 Kdalton polypeptide of lens fiber cells in a calcium-independent manner. Via calmodulin the junctions become calcium-sensitive.

Purified lens junctional protein forms channels in planar lipid films.

Junctions isolated from bovine lenses were solubilized with the detergent octyl glucoside, and their protein(s) was reconstituted in unilamellar vesicles. The protein(s) appears as annular-shaped intramembrane particles approximately equal to 10 nm in diameter on the vesicles' fracture faces. The addition of the vesicle-containing junctional protein(s) to both sides of preformed lipid films induced voltage-dependent channels. The channels have a conductance of 200 pS in 0.1 M salt solutions and are thus large enough to account for the electrical coupling observed between intact lens fibers; they turn off when the magnitude of the voltage is increased and in the presence of octanol. Although the identity of the reconstituted channels as the communicating pathway between lens fibers remains to be proven, it is most likely that the reconstituted channels are formed by MIP-26, the major protein component of the isolated lens junctions.

Functional reconstitution of lens gap junction proteins into proteoliposomes.

Membranes rich in junction complexes were prepared from bovine lens, and the fragments of the membranes were reconstituted into proteoliposomes with a large excess of phosphatidylcholine and dicetylphosphate. The osmotic swelling behavior of these liposomes showed that the lens junction membranes contributed protein components that produced channels with a nominal diameter of 1.4 nm. Most preparations of lens junctions produced rates of osmotic swelling much slower than those found in proteoliposomes containing equivalent amounts of Escherichia coli porin, and we discuss several possible explanations for this observation.

Lens junctional protein: analyzing MP26 with monoclonal antibodies.

Specific antibodies are versatile tools for analyzing cell surface proteins. This study involves the characterization of monoclonal antibodies which are specific for the junctional protein found in the lens fiber cell. This protein can be expected to include regions on the external membrane surface for junction formation, others on the cytoplasmic surface for regulation of junctional properties and, if cell-cell channels are indeed involved, transmembrane domains forming the hydrophilic connection between adjacent cytoplasms. Antibodies to these various regions would provide for an experimental analysis of the junctional protein, e.g., the identification of "active sites" for junction formation. Three monoclonal antibodies specific for the lens junctional protein in the chicken are described here. The first, termed B2, also recognizes the bovine junctional protein, MP26 (5). We have characterized the submolecular specificity of B2 and have found that it binds approximately ten amino acid residues from the C-terminus of MP26. In isolated lens junction preparations, B2 binds to the cytoplasmic surfaces of the lens junctions (both 12 nm and 16 nm thick forms). Thus, we consider MP26 a component of the lens junction. Monoclonal A4, the second antibody considered in detail here, was produced by immunization with lens membranes after treatment with low pH. We have found that lens junctional membranes are separated, or "split," by treatment at pH 2.5-3.0. It appears that A4 binds to the external surface of the junctional membrane; EM studies to confirm this are in progress. In order to map the A4 binding site within the chicken junctional protein and to explore the arrangement of this protein within the membrane, a number of procedures were used to generate fragments of MP26. These included reactions with N-chlorosuccinimide and proteases after acid treatment. Antibody binding to fragments was evaluated with immunotransfer ("Western") procedures. These studies mapped the A4 binding site to the center of the molecule and suggested that MP26 projected externally from the membrane at two different points. These results are consistent with a recent model, based on sequence data (6), for the arrangement of MP26 within the bovine lens membrane.

Lens junctions are communicating junctions

Lens fibers are electrically coupled with each other and directly exchange dyes and metabolites. In most cells, this form of communication is mediated by gap junctions. Lens fibers lack typical gap junctions. The lens junctions, although morphologically similar to gap junctions, differ from them structurally, chemically and immunologically. Nevertheless, recent evidence suggests that indeed lens junctions are communicating junctions. The lens junction protein, MIP26, displays structural characteristics similar to other channel proteins. Once incorporated into liposomes it forms channels permeable to molecules as heavy as 1.5 kDa. Like other communicating junctions, lens junctions assume crystalline arrays and uncouple with Ca++. The liposome incorporated channels close with Ca++ and H+ in the presence of calmodulin (CaM). Partial loss of gating competency occurs after proteolytic cleavage of the C-terminal arm of MIP26. The need for a unique type of communicating junction in lens is unclear. A possibility is that this tissue has some special cell-to-cell transport requirements, in terms of size and/or charge of permeants, not shared by coupled cells of other tissues.

Interaction of calmodulin and other calcium-modulated proteins with mammalian and arthropod junctional membrane proteins

Calmodulin and other calcium-modulated proteins bind in vitro to purified junctional polypeptides from rat liver gap junctions, bovine lens fiber junctions, a chymotryptic fragment from bovine lens junctions, and crayfish hepatopancreas gap junctions. The potential biological relevance of the interaction of calmodulin with junctional proteins is suggested by immunocytochemical localization of endogenous calmodulin in cortical regions of the cell where gap junctions exist. These observations provide a molecular basis for understanding the potential regulatory role of calmodulin on cell-cell communication channels in vivo . In addition, the calmodulin binding represents the first molecular homology that has been found for junctional channel proteins from mammalian and arthropod tissues.

Calmodulin-mediated gating of lens gap junction channels in vesicles

The fiber cells of eye lens communicate directly with each other by exchanging ions, dyes and metabolites. In most tissues this type of communication (cell coupling) is mediated by gap junctions. In the lens, the fiber cells are extensively interconnected by junctions. However, lens junctions, although morphologically similar to gap junctions, differ from them in a number of structural, biochemical and immunological features. Like gap junctions, lens junctions are regions of close cell-to-cell apposition. Unlike gap junctions, however, the extracellular gap is apparently absent in lens junctions, such that their thickness is approximately 2 nm smaller than that of typical gap junctions (Fig. 1,c). In freeze-fracture replicas, the particles of control lens junctions are more loosely packed than those of typical gap junctions (Fig. 1,a) and crystallize, when exposed to uncoupling agents such as Ca++, or H+, into pseudo-hexagonal, rhombic (Fig. 1,b) and orthogonal arrays with a particle-to-particle spacing of 6.5 nm. Because of these differences, questions have been raised about the interpretation of the lens junctions as communicating junctions, in spite of the fact that they are the only junctions interlinking lens fiber cells.

Distribution of the main intrinsic polypeptide in lens fiber membrane domains

The lens fiber cell of the vertebrate eye is a postmitotic, highly differentiated cell which is derived from the lens epithelium. Upon differentiation into lens fibers, the epithelium expresses a unique membrane protein (Main Intrinsic Polypeptide, MIP) with a molecular weight in excess of 26,000. These lens fiber cells are extraordinarily rich in intercellular junctions which can occupy up to 64% of the cell surface. Lens fiber junctions, frequently referred to as “gap junctions”, co-purify with MIP and it has therefore been suggested from biochemical evidence that MIP is a junctional protein.Electron microscopic immunoferritin studies in which rabbit anti-bovine MIP antiserum was incubated with isolated bovine lens fiber membranes have shown that MIP is present in abundance in pentalaminar profiles conventionally regarded as lens junctions (Figure 1).

On the structural organization of biological pumps and channels

The plasma membranes of most animal cells contain transport proteins which function to provide passageways for the transported species across essentially impermeable lipid bilayers. The channel is a passive transport system which allows the movement of ions and low molecular weight molecules along their concentration gradients. The pump is an active transport system and can translocate cations against their natural concentration gradients. The actions and interplay of these two kinds of transport proteins control crucial cell functions such as active transport, excitability and cell communication. In this paper, we will describe and compare several features of the molecular organization of pumps and channels. As an example of an active transport system, we will discuss the structure of the sodium and potassium ion-activated triphosphatase [(Na+ +K+)-ATPase] and as an example of a passive transport system, the communicating channel of gap junctions and lens junctions.

On the structural organization of isolated bovine lens fiber junctions.

Junctions between fiber cells of bovine lenses have been isolated in milligram quantities, without using detergents or proteases. The structure of the isolated junctions has been studied by thin-section, negative-stain, and freeze-fracture electron microscopy and by x-ray diffraction. The junctions are large and most often have an undulating surface topology as determined by thin sectioning and freeze-fracture. These undulations resemble the tongue-and-groove interdigitations between lens fiber cells previously seen by others (D. H. Dickson and G. W. Crock, 1972, Invest. Ophthalmol. 11:809-815). In sections, the isolated junctions display a pentalamellar structure approximately 13-14 nm in overall thickness, which is significantly thinner than liver gap junctions. Each junctional membrane contains in the plane of the lipid bilayers distinct units arranged in a square lattice with a center-to-center spacing of 6.6 nm. Freeze-fracture replicas of the junctions fractured transversely show that the repeating units extend across the entire thickness of each membrane. Each unit is probably constructed from four identical subunits, with each subunit containing a protein of an apparent molecular weight of 27,000. We conclude that the lens junctions are structurally and chemically, different from gap junctions and could represent a new kind of intercellular contact, not simply another crystalline state of the gap junction protein.

The major polypeptide (MIP) of lens fiber junctions and its synthesis in cultured differentiating lens epithelial cells.

Lens and liver contain many gap junctions, which for a long time have been considered to be very similar. Recent results, however, point to differences on morphological and biochemical levels, especially when the liver gap junction polypeptide (26,000 Daltons) is compared with the main intrinsic polypeptide (MIP) from lens junctions. The lens fiber specific MIP, which represents a marker molecule for lens cell differentiation could be detected by indirect immunofluorescence as well as by immunodiffusion in lens epithelial cells, which differentiated in vitro under distinct culture conditions. The fine structure of these differentiated cells is presented.

A Tetragonal Lattice in Mammalian Lens Junctions

A pure fraction of junctional membranes has been isolated in milligram quantities from bovine lens without detergents. SDS-PAGE analysis shows a major band at 27,000 d and a minor one at 21,000 d. Thin section electron microscopy (EM) of this fraction revealed junctions of two closely apposed membranes with an overall thickness of 135-140 Å with a wavy contour and lengths greater than 10 μm. Negative stain and freeze-fracture (FF) EM (only unetched specimens so far) and X-ray diffraction studies of unfixed and unglycerinated specimens were in agreement regarding the structure of the junctions. They consisted primarily of extensive domains of sub-units arranged in a 66 Å tetragonal lattice. Specimens were spun down centrifugally in a Beem capsule and partially dried for the x-ray diffraction studies. The X-ray patterns contained several reflections which indexed on a tetragonal lattice of 66 Å in the equatorial direction parallel to the plane of the membranes and several bands of a continuous transform in the meridional direction consistent with membrane pairs. No diffraction was seen at 85 Å in the equatorial direction.

Gap junction dynamics: reversible effects of divalent cations.

Reversible changes in gap junction structure similar to those previously seen to parallel electrical uncoupling (9, 33, 34) are produced by treating with Ca++ or Mg++ gap junctions isolated in EDTA from calf lens fibers. The changes, characterized primarily by a switch from disordered to crystalline particle packings, occur at a [Ca++] of 5 x 10(-7) M or higher and a [Mg++] of 1 x 10(-3) M or higher and can be reversed by exposing the junctions to Ca++- and Mg++-free EGTA solutions. Similar changes are obtained in junctions of rat stomach epithelia incubated at 37 degrees C in well-oxygenated Tyrode's solutions containing a Ca++ ionophore (A23187). Deep etching experiments on isolated lens junctions show that the true cytoplasmic surface of the junctions (PS face) is mostly bare, suggesting that the particles may not be connected to cytoskeletal elements. A hypothesis is proposed suggesting a mechanism of particle aggregation and channel narrowing based on neutralization of negative charges by divalent cations or H+.